anti-mouse cd8 (53-6.7) pe- cy7  (Thermo Fisher)

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . CD8+ T cells from 5 donors stimulated 3x with pools of 10 peptides, except pool 9 which had 6 peptides. C . 3D modeling of VP1 pentamer highlighting epitopes (Green: VP1 (100-108), Yellow: VP1 (251-259), (253-262), (274-283), Purple: Published epitopes, Blue: Predicted epitopes from IEDB. D . CD8+ T cells from 3 donors, 10 cell lines each, stimulated 3x with pools of peptides and then individual T2 IFN- γ response.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques:

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . Pooled CD8+ T cell lines from 3 healthy donors were split into three different fractions and stained with low, intermediate, or high tetramer concentration. From samples stained with low and intermediate tetramer concentration, live CD3+ CD8+ T cells were sorted from the 90th percentile tetramer(+) gates. Shown here is the cumulative clone count in the 90th percentile gates (y-axis) plotted against maximum enrichment score out of the 11 calculated for each clone (x-axis). Symbol size corresponds to clone count in tetramer-negative gate. Symbol color indicates clone abundance in the single cell TCR dataset (low tetramer concentration condition). High affinity TCRs have high cumulative clone count, high maximum enrichment score, and small symbol size. SumSort = total number in top 10% tetramer binder gate (i.e. 90th percentile) in the 1:2000 + 1:4000 tetramer staining condition. MaxFold = greatest value for fold enrichment (out of the calculated 11). C . Top 14 T cell receptors were virally transduced into human primary T cells in 2 donors. TCR-transduced cells were stimulated with different amounts of VP1(100-108) peptide and tested for intracellular INF- γ content as ratio of positive control (max). EC50 (M) scores populated in tabular format.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Staining, Concentration Assay, Positive Control

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . TCR 3 and TCR 8 tested against K562 presenting VP1(100-108) on HLA-A2 vs a control TCR (DMF5) with its corresponding peptide (MART1) after 48h incubation at unsorted E:T of 0.5 and 2. Error bars repres nt SEM for 3 technical replicates from one representative experiment. p<0.001 by unpaired T test. C . After 72 hours of co-culture with JCV-infected SVG cells and no T cells (no TCR), sham TCR, TCR 3, and TCR 8 looking at live CD8+ T cell CD137 positivity via flow cytometry. TCR 3 and 8 vs no TCR p value <0.001; TCR 3 vs sham TCR+JCV p=0.0032 and TCR 8 vs sham TCR+JCV p=0.029 D . Percent killing of SVG cells determined by flow cytometry based on gating of loss of live astrocytes. p value <0.001; TCR 3 vs sham TCR+JCV p<0.001 and TCR 8 vs sham TCR+JCV p=0.002 by unpaired T test. Done in duplicate at various unsorted E:Ts, mean results shown.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Control, Incubation, Co-Culture Assay, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: Flow cytometry data of live CD8+ T cells stained with HLA-A2:BK VP1 (100-108) and HLA-A2:JV VP1 (100-108) at 1:50 tetramer dilution with an HLA-A2 positive JCV antibody negative healthy donor on the left and an HLA-A2 confirmed PML survivor on the right.

Article Snippet: After incubation, cells were first stained with the LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen L34966), followed by anti-CD8 (PE-Cy7, Invitrogen 25-0087-42) stain.

Techniques: Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . CD8+ T cells from 5 donors stimulated 3x with pools of 10 peptides, except pool 9 which had 6 peptides. C . 3D modeling of VP1 pentamer highlighting epitopes (Green: VP1 (100-108), Yellow: VP1 (251-259), (253-262), (274-283), Purple: Published epitopes, Blue: Predicted epitopes from IEDB. D . CD8+ T cells from 3 donors, 10 cell lines each, stimulated 3x with pools of peptides and then individual T2 IFN- γ response.

Article Snippet: The patient sample was thawed and stained with live dead aqua (Invitrogen L34966), anti-CD8 (PE-Cy7, Invitrogen 25-0087-42), and JCV VP1(100-108):HLA-A*02:01 (PE) (and the bioequivalent BKV VP1(100-108): HLA-A*02:01 (APC) tetramer as described above, followed by analysis on a Fortessa flow cytometer using FACSDiva software (BD).

Techniques:

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . Pooled CD8+ T cell lines from 3 healthy donors were split into three different fractions and stained with low, intermediate, or high tetramer concentration. From samples stained with low and intermediate tetramer concentration, live CD3+ CD8+ T cells were sorted from the 90th percentile tetramer(+) gates. Shown here is the cumulative clone count in the 90th percentile gates (y-axis) plotted against maximum enrichment score out of the 11 calculated for each clone (x-axis). Symbol size corresponds to clone count in tetramer-negative gate. Symbol color indicates clone abundance in the single cell TCR dataset (low tetramer concentration condition). High affinity TCRs have high cumulative clone count, high maximum enrichment score, and small symbol size. SumSort = total number in top 10% tetramer binder gate (i.e. 90th percentile) in the 1:2000 + 1:4000 tetramer staining condition. MaxFold = greatest value for fold enrichment (out of the calculated 11). C . Top 14 T cell receptors were virally transduced into human primary T cells in 2 donors. TCR-transduced cells were stimulated with different amounts of VP1(100-108) peptide and tested for intracellular INF- γ content as ratio of positive control (max). EC50 (M) scores populated in tabular format.

Article Snippet: The patient sample was thawed and stained with live dead aqua (Invitrogen L34966), anti-CD8 (PE-Cy7, Invitrogen 25-0087-42), and JCV VP1(100-108):HLA-A*02:01 (PE) (and the bioequivalent BKV VP1(100-108): HLA-A*02:01 (APC) tetramer as described above, followed by analysis on a Fortessa flow cytometer using FACSDiva software (BD).

Techniques: Staining, Concentration Assay, Positive Control

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: A . Schematic of experimental layout. B . TCR 3 and TCR 8 tested against K562 presenting VP1(100-108) on HLA-A2 vs a control TCR (DMF5) with its corresponding peptide (MART1) after 48h incubation at unsorted E:T of 0.5 and 2. Error bars repres nt SEM for 3 technical replicates from one representative experiment. p<0.001 by unpaired T test. C . After 72 hours of co-culture with JCV-infected SVG cells and no T cells (no TCR), sham TCR, TCR 3, and TCR 8 looking at live CD8+ T cell CD137 positivity via flow cytometry. TCR 3 and 8 vs no TCR p value <0.001; TCR 3 vs sham TCR+JCV p=0.0032 and TCR 8 vs sham TCR+JCV p=0.029 D . Percent killing of SVG cells determined by flow cytometry based on gating of loss of live astrocytes. p value <0.001; TCR 3 vs sham TCR+JCV p<0.001 and TCR 8 vs sham TCR+JCV p=0.002 by unpaired T test. Done in duplicate at various unsorted E:Ts, mean results shown.

Article Snippet: The patient sample was thawed and stained with live dead aqua (Invitrogen L34966), anti-CD8 (PE-Cy7, Invitrogen 25-0087-42), and JCV VP1(100-108):HLA-A*02:01 (PE) (and the bioequivalent BKV VP1(100-108): HLA-A*02:01 (APC) tetramer as described above, followed by analysis on a Fortessa flow cytometer using FACSDiva software (BD).

Techniques: Control, Incubation, Co-Culture Assay, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy

doi: 10.1101/2024.11.04.621904

Figure Lengend Snippet: Flow cytometry data of live CD8+ T cells stained with HLA-A2:BK VP1 (100-108) and HLA-A2:JV VP1 (100-108) at 1:50 tetramer dilution with an HLA-A2 positive JCV antibody negative healthy donor on the left and an HLA-A2 confirmed PML survivor on the right.

Article Snippet: The patient sample was thawed and stained with live dead aqua (Invitrogen L34966), anti-CD8 (PE-Cy7, Invitrogen 25-0087-42), and JCV VP1(100-108):HLA-A*02:01 (PE) (and the bioequivalent BKV VP1(100-108): HLA-A*02:01 (APC) tetramer as described above, followed by analysis on a Fortessa flow cytometer using FACSDiva software (BD).

Techniques: Flow Cytometry, Staining

Journal: Cell reports

Article Title: Unraveling the phenotypic states of human innate-like T cells: Comparative insights with conventional T cells and mouse models

doi: 10.1016/j.celrep.2024.114705

Figure Lengend Snippet: (A) Heatmap showing MetaNeighbor’s AUROC scores between thymocytes split by donor, lineage, and non-effector (c0–11) versus effector (c12–17) clusters. Barplots indicate thymocyte proportions per lineage. (B) Pseudo-bulk differential expression analysis between CD4 + /iNKT and CD8 + /MAIT thymocytes in naive clusters (3, 9, 10, 11). As a negative control, the only three genes that were differentially expressed between CD4 + /MAIT and CD8 + /iNKT thymocytes are displayed in the center of the heatmap. (C) Pseudo-bulk differential expression analysis between CD4 + /CD8 + and iNKT/MAIT thymocytes in naive clusters (3, 9, 10, 11). For both (B) and (C), heatmap displays the expression level of genes (represented with color scale as a Z score of the average normalized expression) that are significantly differentially expressed ( p adj < 0.01). (D) Clustering of hashtag-separated thymic iNKT cells (top), MAIT cells (middle), and γδ T cells (bottom). Right panel shows the score of type I and type III effector gene signature for the corresponding thymic lineage. (E) Kernel density estimates of the normalized expression level of genes of interest. The expression level distribution varies between genes and lineage. The range of kernel density estimate values also varies between each panel (from 0 to 0.04 for the smallest range and 0 to 0.4 for the largest range). A unique color scale was represented to indicate the direction of the values.

Article Snippet: Anti-human CD8ɑ - PE-Cy7 (clone SK1) , Tonbo biosciences , Cat#60-0087; RRID: AB_3106994.

Techniques: Quantitative Proteomics, Negative Control, Expressing

Journal: Cell reports

Article Title: Unraveling the phenotypic states of human innate-like T cells: Comparative insights with conventional T cells and mouse models

doi: 10.1016/j.celrep.2024.114705

Figure Lengend Snippet: (A–C) (A) Clustering of hashtag-separated blood iNKT, MAIT, γδ T, CD4, and CD8 T cells, (B) the respective proportion of cells per cluster and donor, and (C) the effector GEP signature scores (as in ) per cell type and cluster. (D) Top GEP usage for each cell type, based on cNMF-derived usage matrix. (E) Pseudo-bulk, pairwise differential gene expression between cell types. (F) Cell-type-specific genes among T inn cells using GEP5. For both (E) and (F), heatmaps depict the expression level of genes (represented with color scale as a Z score of the average normalized expression) that are significantly differentially expressed ( p adj < 0.01). n = 4, 4, 9, 4, and 9 for iNKT, MAIT, γδ, CD4, and CD8 cells, respectively.

Article Snippet: Anti-human CD8ɑ - PE-Cy7 (clone SK1) , Tonbo biosciences , Cat#60-0087; RRID: AB_3106994.

Techniques: Derivative Assay, Gene Expression, Expressing

Journal: Cell reports

Article Title: Unraveling the phenotypic states of human innate-like T cells: Comparative insights with conventional T cells and mouse models

doi: 10.1016/j.celrep.2024.114705

Figure Lengend Snippet:

Article Snippet: Anti-human CD8ɑ - PE-Cy7 (clone SK1) , Tonbo biosciences , Cat#60-0087; RRID: AB_3106994.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Multiplexing, Recombinant, Antibody Labeling, Staining, Amplification, Software, Flow Cytometry

Journal: medRxiv

Article Title: ATRIP deficiency impairs the replication stress response and manifests as microcephalic primordial dwarfism and immunodeficiency

doi: 10.1101/2024.07.22.24310550

Figure Lengend Snippet: a Total numbers (No) of CD4 + T, CD8 + T, NK, and B cells in the peripheral blood from the ATRIP patient (F1Pt) over time. Shading indicates the age-based reference range. b Flow cytometric (FCM) immunophenotyping of F1Pt, family members, and age-matched healthy controls (HCs). Percentages of CD4 + T, CD8 + T, NK, and B cells in PBMCs from F1Pt, sister (F1Si), mother (F1Mo), and father (F1Fa). Data represents one experiment, with each datapoint representing one biological replicate. Mean and SEM are shown. c IgG2 concentration (Conc.) in the peripheral blood from F1Pt over time. Shading indicates the age-based reference range. Immunoglobulin substation therapy is indicated. d Total numbers (No) of neutrophils in the peripheral blood from F1Pt, demonstrating intermittent neutropenia. Shading indicates the age-based reference range. e Hemoglobulin concentration (Conc.) in the peripheral blood from F1Pt over time. Shading indicates age-based reference range. Corticosteroid (CORT) and anti-CD20 mAb treatment (aCD20) is indicated. f UMAP plot depicting cluster annotation of 25 unique T and NK subsets (left). Analysis was performed using concatenated 25-parameter FCM data of PBMCs obtained from HCs (n = 6) and F1Pt. Contour plots of HCs (middle, top) and F1Pt (middle, bottom). Bar graph showing the relative proportion of HCs and F1Pt within each T and NK subset cluster (right). g UMAP plot demonstrating cluster annotation of 18 unique B and innate subsets (left). Analysis was performed using concatenated 25-parameter FCM data of PBMCs obtained from HCs (n = 6) and F1Pt. Contour plots of HCs (middle, top) and F1Pt (middle, bottom). Bar graph depicting the relative contribution of HCs and F1Pt within each B and innate subset cluster (right). Data is representative of one experiment (f-i). Source data are provided as a Source Data file.

Article Snippet: The cells were seeded in a 48-well plate (250,000 cells in 500 µl), treated with 0.02 µg/ml MMC, and subsequently stimulated with 2% PHA-M. After 96h of culture, PHA blasts were harvested and stained with anti-CD3-AF700 (SK7; BioLegend; 344822), anti-CD4-FITC (RPA-T4; BD Bioscience; 561005), anti-CD8-PE-Cy7 (RPA-T8; BD Bioscience; 557746), and FcR block (BioLegend; 422302) in PBS.

Techniques: Concentration Assay

Journal: medRxiv

Article Title: ATRIP deficiency impairs the replication stress response and manifests as microcephalic primordial dwarfism and immunodeficiency

doi: 10.1101/2024.07.22.24310550

Figure Lengend Snippet: a CITE-seq profiling of PBMCs from healthy controls (HCs) (n =3) and ATRIP patient (F1Pt), identifying 25 immune subsets. UMAP visualization of pooled CITE-seq data of HCs and F1Pt, displaying the identified subsets (left). UMAP plot of HCs (middle, top) and F1Pt (middle, bottom). Ratio of the 25 subsets in HCs and F1Pt, ranked based on the prevalence in F1Pt (right). b Circos plots showing the TRBV and TRAV pairing pattern of CD8 + T effector memory (T EM ) cells of healthy controls (HCs) and ATRIP patient (F1Pt). c Frequency of unique CD8 + T EM cell clones in HCs and F1Pt. d Distribution of the CDR3 region lengths of TCR-α and TCR-β clones from HCs and F1Pt CD8 + T EM cells. e Frequency of class switch recombination junctions per S region in HCs (n = 3) (junctions; n = 8305) and F1Pt (junctions; n = 2758). Mean and SD are shown. f Proportion of non-productive junctions (inversional recombination) per S region in HCs (n = 3) (junctions; n = 652) and F1Pt (junctions; n = 225). Mean and SD are shown. g Pie charts demonstrating the microhomology usage at Sμ-Sα junctions in HCs (n = 3) and F1Pt. Data represents one experiment (a-f). Source data are provided as a Source Data file.

Article Snippet: The cells were seeded in a 48-well plate (250,000 cells in 500 µl), treated with 0.02 µg/ml MMC, and subsequently stimulated with 2% PHA-M. After 96h of culture, PHA blasts were harvested and stained with anti-CD3-AF700 (SK7; BioLegend; 344822), anti-CD4-FITC (RPA-T4; BD Bioscience; 561005), anti-CD8-PE-Cy7 (RPA-T8; BD Bioscience; 557746), and FcR block (BioLegend; 422302) in PBS.

Techniques: Clone Assay

Journal: medRxiv

Article Title: ATRIP deficiency impairs the replication stress response and manifests as microcephalic primordial dwarfism and immunodeficiency

doi: 10.1101/2024.07.22.24310550

Figure Lengend Snippet: a Heatmap representing the top 10 enriched hallmark gene sets (MSigDB) in PBMCs of F1Pt compared to HCs (n = 3). b Volcano plot showing the differentially expressed genes in PBMCs of F1Pt compared to HCs (n = 3). c Heatmap displaying the serum concentration of the cytokines IL-10, IL-21, IL-18, IP-10, IL-6, IL-17A, IL-31, IFNγ, IL-27, MIP31, ICAM-1, IL-1RA, Eotaxin-3, IL-22, and IL-1β in HCs (n = 13) and F1Pt pre- and post-treatment with anti-CD20 mAb. d CD169 and CD64 expression on CD14 + monocytes of HCs (n = 6) and F1Pt pre- and post-treatment with anti-CD20 mAb (aCD20). Histograms of a representative HC and F1Pt are shown. Bar plots depict median fluorescence (MFI). Mean and SEM are shown. e Lymphocyte transformation test (LTT) demonstrating the proliferative reponse of F1Pt whole blood to various stimuli. Data shows at least two independent analyses. f Proliferation analysis of CD8 + and CD4 + PHA blasts from HCs (n = 9) and F1Pt. PBMCs were labeled with CellTrace Violet (CTV) and stimulated with PHA for 96h. Precursor frequency (PF) and proliferation index (PI) of three independent experiments are depicted, with each datapoint representing one biological replicate. Mean and SD are shown. ns: not significant, *p<0.05, ***p<0.001, **** p<0.0001 (multiple unpaired t-tests). Source data are provided as a Source Data file.

Article Snippet: The cells were seeded in a 48-well plate (250,000 cells in 500 µl), treated with 0.02 µg/ml MMC, and subsequently stimulated with 2% PHA-M. After 96h of culture, PHA blasts were harvested and stained with anti-CD3-AF700 (SK7; BioLegend; 344822), anti-CD4-FITC (RPA-T4; BD Bioscience; 561005), anti-CD8-PE-Cy7 (RPA-T8; BD Bioscience; 557746), and FcR block (BioLegend; 422302) in PBS.

Techniques: Concentration Assay, Expressing, Fluorescence, Transformation Assay, Labeling

Journal: medRxiv

Article Title: ATRIP deficiency impairs the replication stress response and manifests as microcephalic primordial dwarfism and immunodeficiency

doi: 10.1101/2024.07.22.24310550

Figure Lengend Snippet: a Schematic outlining the treatment protocol of the flow cytometric (FCM) assay used in b and c . b Cell cycle distributions of untreated fibroblasts from a healthy control (HC) and ATRIP patient (F1Pt). Representative FCM plots are shown (left). Scatter dot plot depicts data from eight independent experiments (right). Mean and SD are shown. ns: not significant (multiple paired t-tests). c FCM EdU pulse-labeling profiles of Mitomycin C (MMC) treated HC and F1Pt fibroblasts (top). Cells were exposed to 0.02 µg/ml MMC for 24h in the absence or presence of an ATR kinase inhibitor (ATRi, 20 nM). Histograms of EdU and DAPI intensity in S phase cells are shown (bottom). The median fluorescence intensity (MFI) is annotated on the histogram. Data is representative of three independent experiments. d Quantification of RPA nuclear fluorescence in HC and F1Pt fibroblasts 3h after 1 mM Hydroxyurea (HU) exposure and concomitant EdU pulse-labeling. The mean RPA intensity per nucleus is shown for EdU+ cells. Dot plot represents data from pooled data from three experiments. The median value is depicted. ns: not significant; ***p<0.001 (multiple Mann-Whitney tests and Bonferroni-Dunn multiple comparisons test). e FCM EdU pulse-labeling profiles of HC and F1Pt fibroblasts after the release from HU treatment (left). Cells were exposed to 1 mM HU for 3h, released for 3h, and subsequently harvested. Histograms of EdU intensity in S phase cells are shown (right). Data are representative of two experiments. f Cell cycle profiles of HC and F1Pt fibroblasts following 72h of 0.02 µg/ml MMC treatment, with and without 20 nM ATRi. The percentage of cells in G2/M phase is indicated. Data is representative of three independent experiments. g EdU pulse-chase kinetics of HC and F1Pt PHA blasts. Cells were untreated or treated with genotoxic inducers (0.02 µg/ml MMC or 200 J/m² UV), pulse-labeled with EdU, and harvested at indicated time points. Kinetic plots show percentages of EdU+ cells present in S phase (middle) and is representative of three independent experiments. h CellTrace Violet (CTV) profiles of CD8 + and CD4 + PHA blasts from HC and F1Pt after 96h of culture in the presence or absence of 0.02 µg/ml MMC. Data is representative of two experiment. Source data are provided as a Source Data file.

Article Snippet: The cells were seeded in a 48-well plate (250,000 cells in 500 µl), treated with 0.02 µg/ml MMC, and subsequently stimulated with 2% PHA-M. After 96h of culture, PHA blasts were harvested and stained with anti-CD3-AF700 (SK7; BioLegend; 344822), anti-CD4-FITC (RPA-T4; BD Bioscience; 561005), anti-CD8-PE-Cy7 (RPA-T8; BD Bioscience; 557746), and FcR block (BioLegend; 422302) in PBS.

Techniques: Control, Labeling, Fluorescence, MANN-WHITNEY, Pulse Chase